Gastrointestinal dysfunction is one of the most common non-motor symptoms in Parkinson's disease (PD). The exact mechanisms behind these symptoms are not clearly understood. Studies in the well-established 6-hydroxydopamine (6-OHDA) lesioned rats of PD have shown altered contractility in isolated circular and longitudinal smooth muscle strips of distal colon. Contractile changes in proximal colon and distal ileum are nevertheless poorly studied. Moreover, segments may serve as better tissue preparations to understand the interplay between circular and longitudinal smooth muscle. This study aimed to compare changes in contractility between isolated full-thickness distal colon muscle strips and segments, and extend the investigation to proximal colon and distal ileum in the 6-OHDA rat model. in vitro organ bath comparing 6-OHDA-lesioned rats with Sham-operated animals. Key Results. Our data showed increased contractility evoked by EFS and methacholine in segments, but not in circular and longitudinal tissue strips of distal colon after central 6-OHDA-induced dopamine denervation. Changes in proximal colon segments were also displayed in high K+ Krebs-induced contractility and spontaneous contractions.  Spontaneous contractions and contractions induced by electrical field stimulation (EFS) and by the non-selective muscarinic agonist methacholine were investigated in strip and/or segment preparations of smooth muscle tissue from distal and proximal colon and distal ileum in an  This study further confirms changes in smooth muscle contractility in distal colon and to some extent in proximal colon, but not in distal ileum in the 6-OHDA rat model of PD. However, the changes depended on tissue preparation. One of the most common non-motor symptoms that significantly affect the quality of life of patients with Parkinson's disease (PD) is gastrointestinal dysfunction, which includes constipation, diarrhea, and fecal incontinence . Cliin vitro. Specifically, we would argue that full-thickness segments include more complex connections with complete mucosal structures and connections between the different layers of the smooth muscle.The mechanisms behind these troublesome non-motor symptoms in PD are poorly understood. The disorder is known to affect both the enteric nervous system as well as the central nervous system , which min vitro organ bath setup in the 6-OHDA rat model of PD. Furthermore, we aimed to explore possible contractile alterations also in the proximal colon and distal ileum. In addition, in an aim to ethically refine the protocols when studying smooth muscle contractility in the 6-OHDA rat model, we also included normal, healthy, animals to investigate if untreated (with non-invasive surgery) animals can replace the Sham-operated control animals.In the current study, we aimed to investigate distal colon contractility using tissue segments and compare this to the more commonly used circular and longitudinal muscle strips, in an ad libitum, and acclimatized for one week before starting the experiments. The study was approved by the local ethical committee . All procedures and animal care were carefully considered to minimize the pain and discomfort of the animals included in the study, and which followed the Directive 2010/63/EU of the European Parliament and of the Council of the European Union (Document 32010L0063).A total of 50 adult male Sprague–Dawley rats , age 8–16 weeks and weighing 260–560 g at the beginning of the experiments, were housed under standard laboratory conditions on a 12 hours/12 hours light/dark cycle. Animals had access to food and water n = 20), or as control by an injection of saline  into the nigrostriatal pathway. Four weeks (25–31 days) post-lesion, the 6-OHDA- and Sham-lesioned animals were sacrificed and tissue samples of the distal and proximal colon, and the distal ileum, were collected for in vitro analysis of smooth muscle function in both circular and longitudinal tissue strips and tissue segments. The brains were fixed by transcardial perfusion with 4% paraformaldehyde (PFA), sectioned, and immunohistochemically stained against dopaminergic neurons using tyrosine hydroxylase (TH) antibodies. In addition to the lesioned animals, untreated rats  were added to further evaluate smooth muscle functions as compared with the Sham-operated controls. The muscle contractility of all animals and tissue samples were evaluated in an isolated organ bath setup using electrical field stimulation (EFS) and direct cholinergic receptor stimulation by the non-selective muscarinic agonist methacholine. In the proximal colon and distal ileum, the EFS- and methacholine-induced contractility was further evaluated in the presence of the cholinergic antagonist atropine. In the distal colon, a subset of animals was evaluated after atropine, whereas a second subset was evaluated in the presence of the nitric oxide (NO) synthase inhibitor L-NAME. Additional segments from the latter animals were inverted inside-out, that is, exposing the luminal space  and evaluated for EFS and methacholine-induced contractility in absence and presence of L-NAME. Spontaneous contractions were finally analyzed in all tissues. The experimental design and timeline are shown in Supplemental The animals were first divided into two groups, which received brain surgery, either by an injection of the 6-OHDA neurotoxin  Krebs solution (“high K+ Krebs”). This revealed unviable responses  in some of the tissue preparations. The EFS data further revealed lack of frequency-dependent responses in a set of the viable tissues. These data, which were evenly distributed through both different groups and respective tissue preparations, were thus excluded from the data analysis. The number of included tissue samples in the Healthy, Sham, and 6-OHDA groups for EFS and methacholine are indicated in The viability of the intestinal tissue was evaluated at start of the −1, AstraZeneca AB, Stockholm, Sweden) was applied before a sagittal incision of ≈1 cm was made on the scalp. A burr hole was further drilled at coordinates, according to Paxinos and Watson  and segments and circular and longitudinal strips were prepared within one hour for functional experiments in the organ bath [Figures μm slices (in five series) with a cryotome . The sections were stored in cryoprotectant (25% ethylene glycol + 25% glycerol in 0.1 MPB) until further immunohistochemistry staining.The 6-OHDA and Sham animals, at 25−31 days post-injection, and the Healthy animals were sacrificed with an overdose of pentobarbital sodium . First, the tissues following mounting were left to equilibrate for 45 minutes at a basal tension of 10–15 mN. The tissue was then challenged with high K+ Krebs (124 mN replacing equimolar amount of Na+ for K+ in the solution) three times to test the viability and activate the tissues. After a washout period of at least 10 minutes and after the tension had returned to baseline, the tissue was challenged with EFS at 1, 2, 5, 10, 20, and 40 Hz . This was followed by a challenge with cumulative concentrations of the cholinergic muscarinic agonist, methacholine . The tissues were further once again challenged with EFS and methacholine at 20 minutes after addition of either the non-selective cholinergic antagonist, atropine (10−6 M) or, for a subset of distal colon segments, the NO synthase inhibitor L-NAME (10−6 M). Washout periods of at least 10 minutes were applied between each set. Finally, a second stimulation with high K+ Krebs solution was performed in each tissue to test the viability at the end of the experiment. The tissue segments and tissue strips were lastly removed from the organ bath, briefly dried, and the wet-weight was measured.The isolated tissue baths contained 20 or 25 mL of Krebs solution aerated with a mixture of 95% O−1) by dividing the absolute contraction with the wet-tissue weight.The contractile responses were recorded and analyzed using the MP100WSW data acquisition system with the Acknowledge Software v4.3 or BSL student v4.1 . The EFS was analyzed by subtracting the mean basal tension, over 10–30 seconds prior to stimulation for each frequency, from the maximum peak value. The response to methacholine was calculated by subtracting the mean peak value for any given concentration and the mean basal tension, over 10–30 seconds, prior to administration of the first concentration. The EFS and methacholine responses were finally calculated as contraction per mg tissue  and 10% methanol in phosphate-buffered saline (PBS), and second pre-incubated for one hour in 5% normal horse serum  and 0.25% Triton-X in PBS. This was followed by an overnight incubation with a mouse anti-TH antibody  in the same solution. Following rinses, the sections were incubated for one hour in biotinylated horse anti-mouse , followed by one-hour incubation in avidin–biotin complex solution . Finally, the staining was revealed by adding 3,3′-diaminobenzidine (Vector Laboratories) and 0.01% H2O2. The sections were mounted on glass slides  and coverslipped using Depex mounting agent . All steps were performed at room temperature with samples kept under continuous motion on a shaking plate , and between each step the sections were washed 3 × 10 minutes with PBS.To identify the DA fibers in the striatum, free-floating sections were immunohistochemically stained against the TH-enzyme as previously described . In briehttp://imagej.nih.gov/ij/). The striatum was outlined as previously described by Carlsson et al. [To estimate the extent of DA lesion in the striatum, the mean optical density at six striatal levels, according to Paxinos and Watson , correspn et al. . Briefly+ Krebs, EFS, and methacholine contractility data are presented as mean ± standard error of the mean (SEM) of the tissue weight-corrected contractions (mN mg−1), or as median with individual data points for the spontaneous contraction data. Statistical analysis and graphs were performed using the GraphPad Prism 9.1.1 for Mac OS . The group- and group-concentration interaction comparisons were done using student's unpaired t-test or two-way Analysis of variance (ANOVA) with Sidak post-hoc test or non-parametric Mann–Whitney U test where appropriate. A p-value less than 0.05 was considered statistically significant. Statistics for significant changes and non-significant trends are presented in respective figure legend. A subset of the data from Figures High Kn = 16; n = 18; unpaired t-test, p < 0.0001; The TH-immunohistochemistry of the striatum revealed that four animals in the 6-OHDA group showed incomplete DA lesion and two in the Sham-operated group displayed a partial DA loss and were therefore excluded from the study. The DA fiber density measurement for the included animals showed an almost complete DA denervation in the 6-OHDA group . The weight of the circular and longitudinal distal and proximal colon tissue strips as well as the distal colon, proximal colon, and distal ileum segments also showed no significant differences between the two groups  were observed in the circular [Figures + Krebs response, a significant increased contractile response to both EFS [−5 M, reached 71–99% inhibition, in the different tissues and groups in the presence of atropine.Following EFS and methacholine no significant differences in contractility , the EFS showed no significant change in either of the groups [The subset of distal colon tissue where the tissue was turned inside out, hence exposing the mucosa (segment inverted) showed, similar as segments, significantly higher EFS-  and methe groups . The effe groups .+ Krebs was observed , the average inhibition of the EFS response reached, at 20 Hz, 61–103% and for the methacholine, at 10−5 M, 63–97%, for all tissues in the Sham and 6-OHDA groups.The 6-OHDA animals showed a trend to increase in tissue weight-corrected response in the circular strips following EFS [Sham: 0.19 ± 0.073 mN mg animals . Followi animals , longitu animals , or segm animals  displaye+ Krebs response were observed in the distal ileum segments between the Sham and the 6-OHDA groups  contractions were observed in the two groups.No changes in high KA groups . Furthert-test, p = 0.580; longitudinal: 2.7 ± 0.8 mN vs. 2.4 ± 0.3 mN, unpaired t-test, p = 0.713; segment: 2.9 ± 0.4 mN vs. 3.3 ± 0.5 mN, unpaired t-test, p = 0.482; for Sham and 6-OHDA, respectively).The spontaneous contraction of the distal colon showed no difference between the Sham and 6-OHDA animals in either of the tissues . The aveU test, p = 0.0133; t-test, p = 0.525; longitudinal: 3.1 ± 0.9 mN vs. 3.6 ± 1.8 mN, unpaired t-test, p = 0.785; segment: 1.9 ± 0.4 mN vs. 1.6 ± 0.06 mN, unpaired t-test, p = 0.354; for Sham and 6-OHDA, respectively).In the proximal colon segments, significant increase in the frequency of spontaneous contraction was evident in the 6-OHDA (median: 35 contractions per 5 minutes) compared with Sham animals [median: 17 contractions per 5 minutes; Mann–Whitney t-test, p = 0.0591; for Sham and 6-OHDA, respectively).The distal ileum segments showed no difference in frequency of spontaneous contractions between the Sham and 6-OHDA groups . A non-s+ Krebs were observed in the Sham group  and Sham (n = 17), respectively; unpaired t-test, p = 0.0141; F = 4.77, p = 0.0399; methacholine: group-concentration interaction, F = 3.61, p = 0.0046). The EFS-induced responses were also lower in the circular strips in this group  = 3.63, p = 0.0057). Similar reduced responses to high K+ Krebs [Healthy (n = 7): 0.85 ± 0.015 mN mg−1vs. Sham (n = 7): 0.47 ± 0.075 mN mg−1; unpaired t-test, p = 0.0422; F = 5.30, p = 0.0400) could also be observed in the circular strips of the proximal colon. In addition, decreased spontaneous contractions  were observed in segments.When comparing the Healthy and the Sham-lesioned animals significant changes in both tissue strips and tissue segments were observed. The alterations were observed both in distal and proximal colon, but not distal ileum. Specifically in distal colon segments, significantly lower responses to high KIn the current study, we have shown the smooth muscle contractility of the distal colon and the proximal colon, but not in distal ileum, are significantly altered following 6-OHDA-induced central DA neurodegeneration at four weeks post-DA lesions. As has been previously reported by others , 13, we 2 receptor expression, which all have been shown to be altered in the distal colon in 6-OHDA rats [This study was not designed to comprehensively study the underlying mechanisms for changes in smooth muscle contractility in different sections of the gastrointestinal tract, but rather to understand differences in different types of tissue preparations. Based on the currently available literature, the observed increase in the cholinergic distal colon contractility is most likely due to a compensatory increase in muscarinic M3 receptor expression, caused by a decreased cholinergic transferase expression and subsequent decrease in release of acetylcholine in the 6-OHDA rat model , 19, 22.HDA rats , 19, 22.NO, apart from being involved in regulation of mucosal secretion, has a prominent role in gastrointestinal motility, acting as an inhibitory neurotransmitter causing relaxation of the smooth muscle tissue –33. Furt+ Krebs solution. These alterations may be caused by previously reported changes in neuronal NO synthase expressing neurons, VIP expressing neurons, as well as TH expression and D2 receptors in the proximal colon of 6-OHDA-lesioned rats [1 antagonists have been shown to reduce peristalsis in the proximal colon [Alterations in contractility were observed in the proximal colon in the current study, mainly in the segments. We unexpectedly did not see any significant changes in EFS- or methacholine-induced responses, but rather changes attributed to significantly increased spontaneous contractility and maximal smooth muscle cell activation by high Kned rats , 9, 22. ned rats . In addial colon . However2 receptors indicate that changes in the ileum in 6-OHDA-leisoned rats do occur [Our data showed no alterations in contractility in the distal ileum segments, indicating that this part of the gastrointestinal tract is not altered regarding contractile function by central DA lesion in the nigrostriatal pathway. However, similar to the proximal colon, histological and neurochemical evidence, including decreased neuronal NO synthase expressing neurons, increase in VIP-reactive neurons and decrease in mRNA expression of Ddo occur , 9, 12. do occur . Togetheper se seems to be of importance. To specifically evaluate changes in gastrointestinal function as a result of DA loss in the nigrostriatal pathway by 6-OHDA, Sham-operated animals are therefore required as controls.Lastly, regarding the aim to understand if Healthy animals could replace Sham-operated animals as control, the current data clearly showed that this are not possible. Several changes in muscle function, in particular the colon, were observed. Thus, the injection in vitro effects and possible role of other neurotransmitters, including DA, serotonin, and noradrenalin, using selective agonists and antagonists in the full-thickness segments of both proximal and distal colon in the 6-OHDA rat model. Once these functions and possible dysfunctions are established, in vivo studies need to be conducted to understand the direct involvement of the central nervous system. This, in turn, hopefully can lead to evaluation of specific drug targets in the gastrointestinal tract related to the characteristic central DA neurodegeneration seen in PD using, among other models, the 6-OHDA rat model of PD.Future aspects from this study involves investigating the in vitro in the proximal colon, but not the distal ileum, following central DA loss caused by 6-OHDA. We believe that the current study sheds further light on the importance of study design and tissue preparations, but also on possible mechanisms of gastrointestinal dysfunction in PD.In conclusion, when studying smooth muscle dysfunction in rats in the 6-OHDA rat model, the choice of tissue preparation should be carefully considered when designing future studies and interpreting results. We suggest, based on the current data, which intact segments may be a better tissue preparation to study. This takes into account the complex interplay between circular and longitudinal contractions. Furthermore, apart from distal colon, alterations in smooth muscle contractility are also evident


Helianthus tuberosus L.) polysaccharide (JAP) is a chain polysaccharide composed of D-fructose connected by β (1-2) glycosidic bonds, which is a kind of inulin. This study evaluated the anti-respiratory syncytial virus (RSV) activity of JAP in vivo and in vitro. To investigate its antiviral activity, an MTT assay, q-PCR, enzyme-linked immunosorbent assay (ELISA), and lung histological observation were performed. The results showed that JAP showed anti-RSV activity in vitro with a half maximal inhibitory concentration (IC50) of approximately 29.15 μg/mL. In vivo results suggested that JAP could effectively inhibit RSV proliferation in the lungs and improve lung tissue lesions in RSV-infected mice. Additionally, JAP could also reduce the expression of TLR3 and TLR4 in the lungs, increase serum anti-inflammatory factors IL-4 levels, and reduce pro-inflammatory factors TNF-α and TNF-β levels, which may be related to its anti-RSV activity. This study provides a new approach to anti-RSV therapy and enriches the potential applications of JAP.Jerusalem artichoke ( Respiratory syncytial virus (RSV), an important RNA virus, belongs to the genus Pneumovirus of the family Paramyxoviridae, which frequently causes lower respiratory tract infections in infants, the elderly, and adults with weakened immunity –4. AccorAlthough the current RSV infection situation is very serious, due to the complex RSV infection mechanism, the different composition of the infected population, and the safety of its use, there is no safe and effective vaccine approved for marketing. The only drugs approved by the US FDA for the prevention and treatment of RSV infection are palivizumab and ribavirin. Palivizumab is a humanized monoclonal antibody, which is mainly used to prevent RSV infection in children with congenital heart disease and high risk of lung disease and premature, and cannot be used for post-respiratory syncytial virus infection treatment . MoreoveLaminaria japonica could exert its anti-RSV activity by up-regulating the production of IFN-α mediated by interferon regulatory factor 3 (IRF3) signaling, with an EC50 of 5.27 mg/mL and a therapeutic index (TI) value of 334 against RSV ) ΔΔCT) method. Specific primers for qRT-PCR are listed in The collected lung tissue was homogenized using a KZ-II homogenizer for RNA extraction to analyze the expression of three target genes RSV, TLR-3, and TLR-4 by qRT-PCR. Total RNA was isolated using Trizol reagent . Reverse transcription reactions were performed using Servicebio® RT First Strand cDNA Synthesis Kit  and Bio-Rad CFX. The reaction was incubated at 25°C for 5 minutes, followed by 30 minutes at 42°C, and finally at 85°C for 5 seconds. Analysis results were normalized to GAPDH. CT values were analyzed using the comparative CT  according to the manufacturer's instructions.The collected blood was centrifuged at 3 000 r/min for 15 min to obtain serum. TNF-P-value of <0.05 was considered statistically significant.Data are expressed as the means ± standard deviation (SD). One-way analysis of variance (ANOVA) for multiple groups was performed using GraphPad Prism 5.0 . A After extraction and purification, the contents of total sugar and reducing sugar in the extract were measured to be 81.93% and 3.83%, respectively. After calculation, the JAP content in the extract was 78.10, and the yield was 11.73%. The JAP obtained in this assay has high purity with the doses of high related values in use.0 of JAP is about 10 mg/mL. According to the values of SI and IC50 index, JAP exhibited a similar inhibitory effect on RSV as ribavirin (The TCibavirin .Body weight change of mice  showed tP > 0.05). More importantly, the weight recovery rate of mice in the treatment group was significantly higher than that of the model group. The mice body weight changes suggested that JAP could significantly improve weight loss in mice induced by RSV infection.The weight of mice in the blank group showed a steady increase. After drug treatment, the body weight of mice in ribavirin and each administration group began to recover on the fourth day, and there was no significant difference between the groups (P < 0.001), suggesting both ribavirin and JAP attenuated RSV-induced lung injury. Moreover, the effect of JAP medium- and high-dose group was better than that in low-dose group, which was comparable to ribavirin from the results of the lung index inhibition rate -dependent and MyD88-independent pathways, thereby inducing various cytokines, and thus induce inflammatory response –25. To fα, TNF-β, and anti-inflammatory factor IL-4 were detected by ELISA to further explore the anti-RSV mechanism of JAP. As shown in α and TNF-β were significantly decreased in the middle- and high-dose groups after 5 days of JAP treatment (P < 0.05). However, the content of IL-4 in serum was only significantly increased in the high-dose JAP group (P < 0.05), which might be related to the dose. It is concluded that JAP might regulate the level of cytokines by inhibiting the levels of TLR3 and TLR4, thereby intervening in the inflammatory response process of infected mice.The serum levels of pro-inflammatory factor TNF-RSV is the main cause of lower respiratory tract infection in infants, the elderly, and the immunocompromised. Furthermore, severe respiratory disease induced by RSV infection is associated with an increased risk of long-term wheezing and later asthma . AlthougJerusalem artichoke, which has the functions of regulating gastrointestinal function, improving immunity, improving lipid metabolism, promoting mineral absorption, lowering blood lipids, and lowering blood sugar [Jerusalem artichoke and evaluated its antiviral activity against RSV in vitro. It was demonstrated that JAP had an anti-RSV SI of 68.27, showing an anti-RSV level comparable to that of ribavirin. Additionally, we also assessed the anti-RSV effect of JAP in vivo. The results suggested that JAP not only significantly improved the weight loss of RSV-infected mice in a dose-dependent manner (JAP is an inulin-type polysaccharide extracted from od sugar –17. Impood sugar , 27–29. t manner  but alsot manner . More imα is an important factor in causing acute inflammatory damage to the lung. IL-4 is recognized to have both anti-inflammatory and immunomodulatory effects. Here, we found that the JAP treatment group could significantly reduce the mRNA levels of TLR-3 and TLR-4, down-regulate the expressions of pro-inflammatory factors TNF-α and TNF-β, and up-regulate the level of anti-inflammatory factor IL-4. The above results suggested that JAP may affect the downstream inflammatory factor levels by inhibiting the expression of TLR3 and TLR4, thereby intervening in the inflammatory response process of infected mice.TLRs are known as the first line of defense against invading pathogens by recognizing and triggering a suitable response against pathogenic attacks, which play an integral role in inflammation, immune cell regulation, survival, and proliferation . A totalJAP, a type of inulin, is a recognized natural prebiotic, which is fermented in the gut to produce a large amount of short-chain fatty acids and significantly increase the number of bifidobacteria, thereby improving the intestinal flora , 33. Furin vitro and in vivo and found that its mechanism involved inhibiting TLR3/4 mRNA levels, down-regulating pro-inflammatory factors TNF-α and TNF-β levels, and up-regulating anti-inflammatory factor IL-4 levels. These data suggest the potential use of JAP as antiviral agent against RSV. However, further studies are needed to elucidate their mechanisms, which remain unclear.In conclusion, we preliminarily confirmed the anti-RSV effect of JAP


The animal experiments also showed that Ng-m-SAIB could promote the osteogenesis of critical size defects in the skull of the osteoporotic model mouse (the senescence-accelerated mouse-strain P6). Together, these results collectively suggested that Ng-m-SAIB might be a promising biomaterial to treat osteoporotic bone defects with favorable osteo-immunomodulatory effects.Repairing osteoporotic bone defects is still a major clinical challenge. Recent studies have revealed that immune response is also essential in osteogenesis. The intrinsic inflammatory response of the host, especially the M1/M2 polarization status and inflammatory secretory function of macrophages, can directly affect osteogenic differentiation. Therefore, in this study, an electrospun naringin-loaded microspheres/sucrose acetate isobutyrate (Ng-m-SAIB) system was constructed to investigate its effect on the polarization of macrophage and osteoporotic bone defects. The results of both   With the aging of society, osteoporosis has become a major health issue, and the treatment of osteoporotic bone defects faces great challenges , 2. The Naringin, a flavonoid, is an important active ingredient in the traditional Chinese medicine’s bone tonic . It can Recent studies have shown that immune response is also an important osteogenesis-associated component , 23. MacIn this study, the Ng-m-SAIB was prepared. It was hypothesized that Ng-m-SAIB could regulate the polarization of macrophages into M2 type and form a microenvironment favorable for osteogenesis, thereby promoting osteogenesis in the osteoporotic bone defects. Therefore, the main objective of this study was to investigate the immunomodulatory functions of Ng-m-SAIB and its osteogenic effects on osteoporotic bone defects.The Ng-m-SAIB and blank microsphere/SAIB (m-SAIB) were prepared as described in our previous study . The preThe RAW264.7 cells (murine monocyte/macrophage cell line) and bone marrow stromal cells (BMSCs) were cultured in a configured high-sugar medium. The components of the cell culture medium were 89% DMEM , 10% fetal bovine serum  and 1% penicillin/streptomycin . Furthermore, the growth medium was replaced every 2–3 days.450) was then measured using a spectrometer .The m-SAIB and Ng-m-SAIB were spread in the center of wells in a 96-well plate and sterilized under ultra-violet (UV) light for 2 h. Then, the RAW264.7 cells were seeded into the wells at a density of 3000 cells/well. After the co-culture with Ng-m-SAIB and m-SAIB for 1, 3, 5 and 7 days, the proliferation activities of the RAW264.7 cells were identified using a Cell Counting kit-8 (CCK8) assay kit . The absorbance  at 450 nm (ODThe prepared Ng-m-SAIB (contains 10 mg Ng-m) and m-SAIB (contains 10 mg blank-m) samples were spread in the center of a 10-cm culture dish and sterilized under UV light for 2 h. A blank dish was used as a negative control group. Approximately 20 000 RAW264.7 cells were added to each culture dish. After co-culture for 2 days, cells were digested and collected. The collected cells were stained using the Annexin V-FITC/PI Apoptosis Detection Kit . Then the fluorescence signals were detected using the flow cytometry  within 1 h after staining was completed, and the FlowJo software was used to analyze the data.The Ng-m-SAIB was spread in the center of 10-cm culture dishes and the material was sterilized under UV light for 2 h. Then the RAW264.7 cells were co-cultured with or without Ng-m-SAIB for 4 days, and then, the RAW264.7 cells were digested and collected.–△△Ct method. The primers used in this study are listed in First, the total RNA from the RAW264.7 cells was extracted using the RNA extraction kit . Then a PrimeScript RT reagent kit  was used to reverse the RNA into cDNA. Quantitative real-time polymerase chain reaction (RT-PCR) was performed with the PrimeScript RT-PCR kit . The RT-PCR reaction proceeded at 95°C for 30 s, 95°C for 5 s and 60°C for 30 s for 40 cycles. Gene expression was calculated with the 2CD206 antibody  and CCR7 antibody , separately, for 1 h. After washing with PBS, the cells were incubated with the respective secondary antibodies  for another 1 h. Finally, the DAPI solution was used to stain the cells for 10 min. The staining results were observed and photographed under a fluorescence microscope.The RAW264.7 cells were co-cultured with Ng-m-SAIB for 4 days and then digested and re-seeded into 24-well plates at a density of 10 000 cells per well, and immunofluorescence staining was performed after the cells were attached to the wells. After the cells were washed three times with Phosphate Buffered Saline (PBS), they were fixed with 4% paraformaldehyde for 20 min and blocked with 5% Bull Serum Albumin for 1 h at room temperature. Then, the cells were incubated with CD206 antibody  or CD86 antibody  at 4°C for 1 h. After 1 h, the excess antibodies were removed by washing with PBS three times. The fluorescence signals were detected using the flow cytometry  within 1 h after staining was completed, and the FlowJo software was used to analyze the data.The RAW264.7 cells were co-cultured with Ng-m-SAIB for 4 days and then incubated with the Ng-m-SAIB were placed in a 10-cm cell-culture dish and sterilized under UV light for 4 h before cell seeding. Then, ∼ 300 000 RAW264.7 cells were co-cultured into each culture dish for 4 days. Then, the cell culture medium was collected, and the sediment was removed by centrifugation. The supernatant was then configured with a normal medium in a ratio of 1:2 to form a conditioned medium, which was stored at –80°C until further use.The BMSCs were seeded into a 96-well plate, and the density was 1000 per well. On the second day, the experimental group replaced the cell culture medium with a conditioned medium. Then, on Days 1, 3, 5, and 7 of changing the medium, the cell counting was performed using CCK8 assay as described previously in Section ‘Cell counting kit-8 assay’.The BMSCs were cultured in the normal and conditioned media, respectively. Alkaline phosphatase (ALP) staining was performed on Days 7 and 14. The cells were fixed in 4% paraformaldehyde for 30 min at room temperature, followed by washing with PBS. Then the appropriate amount of the configured staining solution was added according to the instructions of the BCIP/NBT Alkaline Phosphatase Color Development Kit . The staining results were observed and photographed under a light microscope .The BMSCs were cultured in normal and conditioned media and then stained with alizarin red staining at Day 21. After rinsing three times with PBS, the BMSCs were fixed in 4% paraformaldehyde. Then, appropriate alizarin red staining solution was added  to stain the cells for 1 h. The calcium nodules were dissolved by adding 10% cetylpyridinium chloride solution  for the semi-quantitative analysis, and the microplate reader  was used to test the optical density at 562 nm.The BMSCs were collected after culturing in two different media for 14 days. Then, follow the method described in Section ‘Quantitative real-time polymerase chain reaction’. The primers used for this experiment are listed in n = 6), including control, m-SAIB, Ng-SAIB and Ng-m-SAIB groups. All the mice were anesthetized by isoflurane inhalation using a small animal anesthesia machine . After dehairing the top of the mice’s heads, the surgical site was disinfected, and a longitudinal skin incision was made using a scalpel to expose the bone surface. Then, a 3-mm full-thickness bone defect was made by low-speed drilling and saline cooling on both sides of the cranial parietal bone. The bone defects were placed with m-SAIB, Ng-SAIB, Ng-m-SAIB or no material. Their skins were sutured at the end of surgery.A total of 24 male senescence-accelerated mouse-strain P6 (SAMP6) mice (16 weeks of age with an average weight of 25–30 g) were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center  and randomly divided into four groups . The cranial parietal bones of all the mice were removed intact and immersed in 4% paraformaldehyde for fixation. The hearts, livers, spleens, lungs, kidneys and brains of the mice in control and Ng-m-SAIB groups were also collected after 8 weeks.After 4 and 8 weeks of surgery, the mice in each group were sacrificed using Automated COF4/80 antibody , iNOS antibody , CD206 antibody , TNFα antibody  and IL10 antibody . After staining, a fluorescence microscope  was used to observe and photograph the sections. The average fluorescence intensity of each sample in the images was analyzed using the ImageJ software.First, the skull samples were decalcified by immersing them in a test tube, containing the ethylene diamine tetraacetic acid solution . The test tube was then placed at 37°C in a shaking incubator. The decalcifying solution was refreshed every 2 days to speed up the decalcification process. The skull specimens were paraffinized after decalcification and sectioned. After dewaxing the paraffin sections, the immunofluorescence staining was performed as described in the literature . The folThe computed tomography (CT) scans of all cranial specimens were performed using the micro-CT . The 3D image reconstruction was performed using the accompanying SCANCO analysis software, and the bone volume-to-total volume ratio (BV/TV) and bone mineral density (BMD) of the defect sites were identified.in vivo’. After dewaxing and hydrating, the paraffinized sections were stained with a hematoxylin–eosin (H&E) staining kit  and a Masson staining kit , following their respective manufacturer’s instructions. The ALP immunofluorescence staining was performed as described previously [The skull specimens were paraffinized as described in Section ‘Effects of Ng-m-SAIB on the polarization of macrophages eviously  using thThe individual organs of the mice in both groups (as described in Section ‘Sample harvesting’) were fixed and paraffinized. The tissue sections were stained with the H&E staining kit  and the tissue morphological changes were observed.P < 0.05 and **P < 0.01, respectively).The data were expressed as mean ± standard deviation. All the data analyses were performed using SPSS 20.0 software . The differences between the groups were analyzed using analysis of variance and Student–Newman–Keuls tests. The significant and highly significant differences were represented by one and two asterisks  and TNFα (pro-inflammatory) secreted by macrophages under the action of Ng-m-SAIB using RT-PCR analysis. The results showed that Ng-m-SAIB could specifically enhance the expression level of IL10 and reduced that of TNFα in vitro. Moreover, the immunofluorescence staining results also showed that the Ng-m-SAIB group had the highest proportion of F4/80-positive cells in the cranial defects at both the time point (after 4 and 8 weeks) in vivo, and the relative levels of CD206 and IL10 were also the highest. These results indicated that Ng-m-SAIB could recruit more macrophages, promote the polarization of macrophages toward M2 and induce the secretion of anti-inflammatory factors.In the current study, Ng-m-SAIB was prepared. Both the ogenesis , 44, 45.in vitro results demonstrated the indirect ‘immune osteogenic’ effects of Ng-m-SAIB.To further verify the effects of cytokines secreted by Ng-m-SAIB-polarized macrophages on the osteogenic differentiation, the conditioned medium was prepared by directly co-culturing the Ng-m-SAIB with macrophages, and the BMSCs were incubated with the conditioned medium. The CCK8 assay results revealed that the conditioned medium promoted the proliferation of BMSCs, and the alizarin red and ALP staining results and RT-PCR results confirmed that the conditioned medium promoted osteogenic differentiation. In conclusion, these However, there are still some things that could be improved in this experiment. Numerous factors might affect the polarization of macrophages. Besides the drug contained in the material, the particle size and shape of the material, pore size and porosity and material size and roughness might also affect the results , 34, 45.Osteoporosis has been recognized as a progressive systemic disease. It is characterized by a reduction in bone mass and changes in the bone microstructure, which might likely cause fragility fractures . The ostOCN and Runx-2 [Naringin has the dual effect of promoting osteogenesis and inhibiting osteolysis, so it can be used to treat osteoporosis . Ng-m-SAd Runx-2 . Studiesd Runx-2 . Moreoved Runx-2 , 51. In In vitro experiments in this study showed that Ng-m-SAIB could promote the polarization of macrophage toward M2 and secrete cytokines favorable to osteogenesis, achieving an indirect immune osteogenic effect. Therefore, we also conducted animal experiments to investigate its effect on promoting osteoporotic bone defects in vivo.However, naringin has not only direct action on bone cells as previously described. The SAMP6 mouse is an ideal animal model to study age-related osteoporosis. Numerous studies have confirmed that SAMP6 mice have essentially similar characteristics to those of human age-related osteoporosis . Our prein vivo results confirmed the osteogenesis-promoting effects of Ng-m-SAIB in the osteoporotic bone defects in comparison with the control, m-SAIB and Ng-SAIB groups.The ability of numerous biomaterials to promote osteogenesis in osteoporotic bone defects has been recently demonstrated. Although the alendronate-loaded scaffold developed by Zeng et al. promoted the repair of cranial defects in osteoporotic SD rats, the problem of burst drug release from this scaffold still requires further improvement . Chu et in vivo experiments was selected based on the results of in vitro experiments and the dosage used in previous literature [in vivo osteogenesis requires further investigation. The osteogenic observation period for in vivo experiments can be further clarified based on the results of subsequent in vivo release and degradation of Ng-m-SAIB.In this study, the dose of naringin used in terature , 60–62. terature , 63. TheIn summary, Ng-m-SAIB is a sustained drug delivery system having good biocompatibility. It can promote the polarization of macrophages toward M2, thereby forming a favorable immune microenvironment and achieving the ‘immune osteogenesis’ effect. Also, Ng-m-SAIB has ‘direct osteogenesis’ effects on the repair of osteoporotic bone defects. Although the specific mechanism of cytokines secreted by macrophages in osteogenesis remains to be further investigated, the experimental results of the present study still demonstrate that Ng-m-SAIB might be a promising biomaterial having the potential of promoting osteogenesis in osteoporotic bone defects.rbad006_Supplementary_DataClick here for additional data file.


Type 2 diabetes mellitus (T2D) is associated with an increased fracture risk. There is little evidence for the effects of glucagon-like peptide 1 receptor agonists (GLP-1RA) on fracture risk in T2D. We aimed to investigate the risk of major osteoporotic fractures (MOF) for treatment with GLP-1RA compared to dipeptidyl peptidase 4 inhibitors (DPP-4i) as add-on therapies to metformin.We conducted a population-based cohort study using Danish national health registries. Diagnoses were obtained from discharge diagnosis codes (ICD-10 and ICD-8-system) from the Danish National Patient Registry, and all redeemed drug prescriptions were obtained from the Danish National Prescription Registry (ATC classification system). Subjects treated with metformin in combination with either GLP-1RA or DPP-4i were enrolled from 2007 to 2018. Subjects were propensity-score matched 1:1 based on age, sex, and index date. MOF were defined as hip, vertebral, humerus, or forearm fractures. A Cox proportional hazards model was utilized to estimate hazard rate ratios (HR) for MOF, and survival curves were plotted using the Kaplan-Meier estimator. In addition, Aalen’s Additive Hazards model was applied to examine additive rather than relative hazard effects while allowing time-varying effects.In total, 42,816 individuals treated with either combination were identified and included. After matching, 32,266 individuals were included in the main analysis . Median follow-up times were 642 days and 529 days in the GLP-1RA and DPP-4i group, respectively. We found a crude HR of 0.89 [0.76–1.05] for MOF with GLP-1RA compared to DPP-4i. In the fully adjusted model, we obtained an unaltered HR of 0.86 [0.73–1.03]. For the case of hip fracture, we found a crude HR of 0.68 [0.49–0.96] and a similar adjusted HR. Fracture risk was lower in the GLP-1RA group when examining higher daily doses of the medications, when allowing follow-up to continue after medication change, and when examining hip fractures, specifically. Additional subgroup- and sensitivity analyses yielded results similar to the main analysis.In our primary analysis, we did not observe a significantly different risk of MOF between treatment with GLP-1RA and DPP-4i. We conclude that GLP-1RA are safe in terms of fracture. Although bone mineral density (BMD) is normal or even elevated in individuals with type 2 diabetes mellitus (T2D), T2D has been associated with an increased fracture risk . In addiGlucagon-like peptide-1 receptor agonists (GLP-1 RA) and dipeptidyl peptidase 4 inhibitors (DPP-4i) were both introduced in Denmark in 2007, and new drugs in the classes are continually being introduced . GLP-1 RKnowledge about the impact of GLP-1 RAs on bone health and fracture risk is limited. Studies attempting to investigate the effects of various glucose-lowering drugs on fracture risk are often subject to confounding and insufficient follow-up durations . Cohort For DPP-4is, most studies reported no association with fracture risk –25. HoweIn the present study, we aimed to investigate fracture risk in individuals using GLP-1 RAs versus individuals using DPP-4is. We hypothesized that there is no difference in fracture risk between the two drug classes.The STROBE guideline for reporting of observational studies was followed  were matched 1:1 using propensity scores.We conducted a nationwide registry-based cohort study using data from the Danish national registries. We included all individuals who initiated a combination of metformin and GLP-1 RA or metformin and DPP-4i treatment between January 1Danmarks Statistik, project identifier no. 703382). Statistics Denmark obtained data from national Danish registries. All Danish citizens are assigned a unique 10-digit personal identification number (PIN) stored in the Danish Civil Registration System, which contains high-fidelity individual-level information on all residents in Denmark and Greenland  from 1977 until 1993 and according to ICD-10 from 1994 onwards.All data were provided in anonymized form by Statistics Denmark . For each medication, we defined a start date (date of first redemption) and an end date (date of last redemption plus the number of daily doses redeemed on that date). We then excluded all individuals in which treatment with GLP-1 RAs and DPP-4is overlapped for the entire duration of treatment and those in which neither medication overlapped with metformin use. Remaining individuals were assigned to the exposure or control group based on which medication was first taken singularly in combination with metformin.We first identified persons treated with metformin and GLP-1 RAs (the exposure drug) and/or DPP-4is (the control drug) between January 1End of combination therapy was defined as the day that treatment with metformin, the exposure drug, or the control drug ceased or when another glucose-lowering drug was initiated. Glucose-lowering drugs were defined as any drugs with an ATC code beginning in “A10”; i.e., insulins and analogues, biguanides, sulfonylureas, alpha-glucosidase inhibitors, thiazolidinediones, DPP-4is, GLP-1 RAs, sodium-glucose co-transporter 2 inhibitors, and repaglinide.Then start and end dates were defined for each other class of glucose-lowering medication. Those who were already treated with an additional glucose-lowering drug  at the beginning of combination therapy were included if (and when) the additional medication was halted and the individual thus received only a combination of metformin and GLP-1 RA or metformin and DPP-4i treatment. beginning of combination therapy was between January 1st 2007 and Dec 31st 2018.Finally, the cohort was limited to those in which The National Prescription Registry contains data on redeemed drug prescriptions along with dates, doses and pack sizes. Each medication–including the exposure and control medications–was only considered used if an individual had redeemed at least three prescriptions in the period outlined above. Medications were identified using ATC codes  variable, which is based on “the assumed average maintenance dose per day for a drug used for its main indication in adults”, according to the World Health Organization Collaborating Centre for Drug Statistics Methodology . The resOf note, exposure to metformin, the exposure drug, and the control drug was assumed to be continuous between initiation and end-of-treatment. To estimate the effects of pauses in these drugs, we calculated the cumulative dose  for each drug between the last prescription redeemed prior to or at index date until end of follow-up for each individual. We then assessed pauses using the medication possession ratio (MPR); the ratio of the cumulative number of daily doses to the number of days in the same period. To remove the effects of pauses in medication or low average medication dose, several thresholds for MPR were used: MPR ≥ 0.5, MPR ≥ 0.75, and MPR ≥ 0.95. Lower thresholds likely exclude individuals without pauses in medication, whereas higher thresholds more likely relate to the actual dosage that individuals receive .end of combination therapy, emigration, death, or December 31st 2018, whichever came first.The follow-up period was defined as the time between the index date and The primary outcome was incident major osteoporotic fracture (MOF). MOF were defined as any of the following fractures: Hip, vertebral, humerus, or forearm fracture. Fractures were identified by ICD-10 codes (Data on covariates were obtained using ICD-8 (1977–1993) and ICD-10 (1993–2018) codes .As a proxy for heavy smoking (binary variable), we used diagnosis codes related to lung diseases highly associated with tobacco exposure along with diagnosis codes related to nicotine or tobacco, previous use of medications for the treatment of tobacco dependence, and initiation of drugs for obstructive airway disease after the age of 40.Obesity, alcohol consumption and hypertension (binary variables) were defined by any diagnosis codes related to the conditions in question and/or ever use of medications for their treatment.Late-diabetic complications, inflammatory bowel disease (IBD), kidney disease, and previous falls (binary variables) were identified through diagnosis codes.The Charlson Comorbidity Index  was calculated based on other comorbidities. The CCI was modified to exclude kidney disease and late-diabetic complications, as these covariates were separately adjusted for in the statistical analyses.Previous insulin use and previous glucocorticoid use were identified through redeemed prescriptions (binary variables).SOCIO13 classification) were identified on the year preceding each individual’s index year. Income  was adjusted for inflation to a 2018 level according to the Consumer Price Index provided by Statistics Denmark and converted from DKK to Euros using an exchange rate of 7.4363 DKK/Euro.Income (numeric variable) along with marital status and employment status  were also calculated for all baseline variables as recommended for propensity-score matched studies (Descriptive statistics are presented as numbers and proportions (%), means and standard deviations (SD), or medians and interquartile ranges (IQR). In the case of CCI, median and 10 studies . Cohen s studies , 37.There were only missing data in the socioeconomic variables . Income was used as a covariate in the main analysis, and missing data were imputed beforehand. Missing data were assumed to be missing at random, and multiple imputation was performed by multivariate imputation using chained equations , 39. TenDue to imbalances in sex, age at baseline, and inclusion date, we matched the two groups on propensity scores estimated from these variables. A binomial logistic model was fitted to age, sex, and inclusion date using treatment group as the dependent variable , 41 and We matched subjects 1:1 on the logit transformation of the propensity score by nearest-neighbor (“greedy”) matching without replacement, using a caliper width equal to 0.2 x the (pooled) SD of the transformed propensity scores , 43.For multiple imputed datasets, matching and statistical analyses were performed separately on each resultant dataset, and the statistical estimates were finally pooled.k:1 matching was performed, with k being the highest possible number up to 10 which allowed every individual in the exposure group to be matched to k controls within the set calipers.For subgroups, matching was done using the previously computed propensity scores. In the subgroups examining specific GLP-1 RAs, After matching, balance in the matched variables was assessed by inspecting the distributions of propensity scores across groups and by calculating SMDs for each matching variable.Multicollinearity was assessed using the Variance Inflation Factor (VIF) which yielded values no higher than 1.4 for any covariate. In addition, we examined Pearson’s partial correlation coefficient for each pair of variables, and none revealed significant correlations.On a non-imputed matched dataset, the Kaplan-Meier Estimator was used to produce survival plots for all fracture types; a survival plot for MOF on a non-matched dataset was also produced . For eacFor the primary analysis, we used the Cox proportional hazards model to estimate hazard rate ratios (HRs) for fracture between the exposure and the control groups. We estimated both crude and adjusted HRs for primary and secondary outcomes. The proportional hazards assumption was evaluated by examining the scaled Schoenfeld residuals of each variable . In the Finally, to examine a possible additive effect of GLP-1 RAs on fracture risk, we used Aalen’s additive hazards regression model; that is, to examine whether absolute rather than relative differences in hazard could be found . In shorSeveral sensitivity and subgroup analyses were performed.First, we examined males and females separately. Second, we performed sensitivity analyses excluding those with low MPR (selected thresholds are described previously in the section Exposure) in either metformin or study drug (GLP-1 RA or DPP-4i) during the study period. Third, we examined a cohort excluding individuals with kidney disease, previous pancreatitis, or previous falls. Fourth, we performed a sensitivity analyses excluding individuals with follow-up times less than 6 months. Fifth, we split the GLP-1 RA group into specific drug groups–liraglutide, semaglutide, exenatide, dulaglutide, and lixisenatide–based on the drug of which they had received the largest cumulative dose during the study period; ties were handled by allowing any person to appear in several of these subgroups, and only three persons did so. Sixth, we performed the main analysis in the full cohort without prior matching. Seventh, we performed a sensitivity analysis excluding individuals treated with systemic glucocorticoids within the last year prior to inclusion, while not allowing follow-up to continue past initiation of systemic glucocorticoid treatment. Lastly, we performed an analysis analogous to the “intention-to-treat” approach in clinical trials; we continued follow-up after changes in medication for an extra 2 years – or until death or emigration, whichever came first. The sensitivity and subgroup analyses were performed on matched groups unless stated otherwise.All analyses were performed using R 4.1.0  in the integrated development environment (IDE) RStudio 1.4.1106 . For imputation, the package “mice” (v 3.13.0) was used. Matching was performed using “MatchIt” (v. 4.2.0) and, for multiply imputed datasets, “MatchThem” (v. 1.0.0). Survival analyses–i.e., Cox model, Kaplan-Meier estimator, and Aalen’s additive hazards regression–were performed using packages “Survival” (v. 2.1.11), “Survminer” (v. 0.4.9), and Survey (v. 4.0).We identified 42,816 subjects treated with metformin in combination with either GLP-1 RAs  or DPP-4is . After propensity-score matching, a total of 32,266  remained.Median [IQR] follow-up times in the two groups were of 642  days in the GLP-1 RA group and 529  days in the DPP-4i group. In total, we had 75,848 years of combined follow-up time.th-90th percentile] of 0 [0–2] in both groups. Previous MOF were equally prevalent (9.6%) in both groups.Sex was balanced between the groups with 42.3% females in the GLP-1 RA group vs. 41.3% in the DPP-4i group. The GLP-1 RA group had a mean ( ± SD) age of 57.5 ( ± 11.3) vs. 57.9 ( ± 11.0) years in the DPP-4i group. Median [IQR] diabetes duration was longer in the GLP-1 RA group with 4.95 [2.15–8.55] years compared to 3.80 [1.33–7.03] years in the DPP-4i group. CCI scores were balanced with medians , which was unaltered in the fully adjusted model (HR 0.65 [0.46–0.93]). The crude HR for vertebral fractures was 0.70 [0.46–1.07] with no change after full adjustment (HR 0.71 [0.46–1.11]) when comparing GLP-1 RAs with DPP-4is. For the humerus, the crude HR was 0.92 [0.68–1.24], and the adjusted HR was 0.91 [0.66–1.25] when comparing GLP-1 RAs with DPP-4is. Estimates for any fracture and for forearm fractures were neutral; for forearm fracture the crude HR was 1.12 [0.85–1.47] and the fully adjusted HR 1.06 [0.79–1.41], and for any fracture the crude HR was 1.01 [0.90–1.13], and the fully adjusted HR was 0.97 [0.86-1.09] when comparing GLP-1 RAs with DPP-4is.Subgroup and sensitivity analyses are presented in Effects between groups were similar between males and females. No changes in effect sizes were observed when excluding individuals with chronic kidney disease, previous pancreatitis and previous falls. When examining different thresholds for MPR, a clear trend was apparent with larger difference in fracture risk for increasing MPR thresholds between the GLP-1 RA group and the DPP-4i group. At MPR ≥ 0.5, the adjusted HR was quite similar to that in the main analysis (HR 0.87 [0.71-1.06]), but this became lower at MPR ≥ 0.75 (HR 0.75 [0.60 – 0.95]) and lower yet at MPR ≥ 0.95 (HR 0.62 [0.46 – 0.84])When excluding subjects with follow-up times shorter than 6 months, 25,390 individuals remained, and the unadjusted HR for MOF was found to be 0.86 [0.73–1.02] for GLP-1 RAs compared to DPP-4is. The fully adjusted model yielded a similar HR of 0.84 [0.71–1.00].Dividing the GLP-1 RA group into subgroups based on the specific drug yielded five groups; liraglutide, semaglutide, exenatide, dulaglutide, and lixisenatide. However, liraglutide users comprised the far majority of GLP-1 RA users (92%), and no other subgroup had sufficient fracture rates to allow reasonable estimation of HRs.Examining the full (unmatched) cohort for MOF risk yielded a significant protective effect in the GLP-1 RA group (unadjusted HR 0.67 [0.58–0.78]) compared to the DPP-4i group. The same effect can be seen in the Kaplan-Meier plot of MOF in the unmatched cohort (p < 0.0001). However, this effect was attenuated in the fully adjusted model to entirely resemble the matched analyses (HR 0.87 [0.74–1.02]).The results were not altered when defining recent or ongoing glucocorticoid use as an exclusion criterion (adjusted HR for MOF 0.89 [0.74–1.08]). When performing an “intention-to-treat” analysis, the HR for MOF was found to be 0.89 [0.78–1.02] with GLP-1 RAs compared to DPP-4is, although this became significant in the fully adjusted model (HR 0.85 [0.74 – 0.98]).intention-to-treat analysis revealed a continuing downward slope for GLP-1 RAs and a less pronounced attenuation on intermediate time-scales .Using Aalen’s additive hazards regression model, we attempted to model the effects of the drugs on fracture in an entirely different way Figure 3As a final measure, we analyzed deaths in the two groups in order to assess potential bias induced by an imbalance in these. In the GLP-1 RA group, 190 (1.2%) deaths occurred with a median [IQR] time-to-event of 647  days, whereas the DPP-4i group experienced 175 (1.1%) deaths with a median time-to-event of 549 [230–1099] days. Indeed, the crude HR for death (with MOF as a censoring event) in the GLP-1 RA group with the DPP-4i group as reference was 0.94 [0.77–1.15]. When adjusted for age and sex, this became 0.96 [0.78–1.18] and when fully adjusted 0.78 [0.64–0.98].In addition to the estimates of treatment on fracture risk, we have presented all covariate estimates from the main analysis of MOF in In the present study, we found that the risk of MOF was slightly lower, albeit not significantly, in those treated with GLP-1 RA compared to those treated with DPP-4is as add-on therapies to metformin. HRs were generally on the order of magnitude of 0.85-0.90; i.e., a 10-15% lower risk of fractures with GLP-1 RAs. These results were similar across various analyses, which will be summarized in the following.Examining specific fracture sites revealed non-significantly reduced risk of fractures of the humerus and of the spine with GLP-1 RAs compared to DPP-4is. Interestingly, however, in the case of hip fractures, we found a statistically significant effect of GLP-1 RAs compared to DPP-4i with risk reductions of as much as 30-35%. Risks of any fracture and of forearm fracture were similar between the groups.When estimating HRs for MOF in the full unmatched cohort, the unadjusted analysis yielded a highly significant difference between the groups. However, this higher risk in the unmatched DPP-4i group appeared to be confounded by age, as the unmatched DPP-4i group was on average 7 years older than the GLP-1 RA group; indeed, the effect was attenuated in the adjusted analyses to resemble the results of the main analysis.In our “intention-to-treat” sensitivity analysis with an additional two years of follow-up, we found an unadjusted HR very similar to the main analysis, although this became a significant protective effect in the fully adjusted analysis. Since changes in bone tissue manifest as fractures with a long delay, this may hint at more pronounced slow-acting effects on bone of the two drugs, although imbalances in confounding factors between the groups may also arise as time passes.Similarly, when increasing the minimum thresholds for average daily dose received in the analysis of MOF, differences between the two groups became larger and increasingly significant. Although this study was not designed to examine a dose-response relationship between the exposure and the outcome, this finding may indicate a dose-dependent effect. This lends credence to a causal interpretation of the associations discussed above.As increased fall risk may be a contributor to the fracture risk in diabetes , we perfIn order to rule out differential mortality as a source of bias in our study, we estimated HRs of death in the two groups and found a negligible difference, although this became significant in the fully adjusted model. A lower mortality in the GLP-1 RA group would expectedly lead to an underestimation of the fracture risk in that group, thereby exaggerating a protective effect of GLP-1 RAs. However, due to the small number of deaths, we believe that the magnitude of such an effect must be negligible.Observational studies and meta-analyses of RCTs on fracture risk with GLP-1 RAs have found mostly neutral effects , 14, 50,However, studies on markers of bone health point towards direct beneficial effects of GLP-1 RAs on bone. Two randomized controlled trials demonstrated reduced bone loss during weight loss with GLP-1 RA compared to placebo , 52. IndThis cohort study was performed using data from Danish nationwide registries. These contain individual-level data on all prescription medications and diagnosis codes along with socioeconomic factors. This provides high-fidelity information on diseases and treatments in the whole period in which GLP-1 RAs and DPP-4is have been marketed in Denmark, allowing an unbiased study population with very little missing data, and providing results which are highly generalizable to other similar populations.The use of DPP-4is as a comparator provided a highly comparable control group, particularly as both drugs were used in the setting of sole add-on medication to metformin, and both drugs have similar priority in the management of T2D. However, GLP-1 RAs are often preferred for T2D subjects with obesity or cardiovascular disease, providing a potential for confounding by indication. Although we attempted to adjust for this, we did not have direct measurements of BMI. In addition, a large proportion of the GLP-1 RA group (13.8%) had received DPP-4is before baseline, whereas only 0.8% of the DPP-4i group had received GLP-1 RAs prior. This indicates that DPP-4i treatment is in some cases attempted before switch to GLP-1 RAs, as the cost of DPP-4is is lower, and GLP-1 RAs (during the period in which this study was conducted) required injections. The price difference between the drugs was reflected in the income gap between the two groups, which was however diminished with matching. The tendency for some individuals to have received DPP-4is before switching to GLP-1 RAs may account for the longer diabetes duration and the slightly higher prevalence of diabetic complications and hypertension in the GLP-1 RA group.Propensity-score matching is a method of mimicking some of the characteristics of a randomized controlled trial , 42, andIn addition, a variety of subgroup and sensitivity analyses confirmed the finding from the main analysis. This supports the conclusion of neutral or slightly reduced risk of fracture with GLP-1 RA treatment compared to DPP-4i treatment in this population.Residual confounding in an observational study cannot be ruled out. Particularly, we were unable to account for diet and exercise, both of which may serve as confounders. Lack of access to lab results and other clinical information prevented adjustment for variables such as BMD, BMI, and glycemic control . In addition, some covariates such as smoking and alcohol consumption were crudely estimated through diagnosis codes and previous medications. Similarly, the utility of diagnosis codes to identify falls and other risk factors for fracture is limited, and therefore differential fall patterns between the two groups may still be a cause of residual confounding. However, those treated with GLP-1 RAs appear to have higher prevalence of late-diabetic complications and previous SU and insulin use. These factors indicate that the GLP-1 RA group is more severely affected by diabetes compared to the DPP-4i group, and the GLP-1 RA group may thus be subject to residual confounding associated with higher fracture risk; this would in turn lead to over-estimation of fracture risk in those receiving GLP-1 RAs. As a consequence, the true HR would potentially be more in favor of GLP-1 RAs than the HRs observed in this study.As changes in bone structure take time to manifest as fractures, median follow-up times less than 2 years may not be sufficient to fully assess the effects of these drugs. However, in the matched cohort, a full 13,767 individuals had more than two years of follow-up time, with nearly half of those  having more than four years.In our primary analysis, the risk of MOF was not significantly different between users of GLP-1 RA and DPP-4i. However, in a secondary analysis, users of GLP-1 RA exhibited a significantly lower risk of hip fracture and a lower risk of MOF compared to DPP-4i users when allowing follow-up to continue after medication change. In addition, when examining higher doses of treatment, the difference in MOF risk between the two groups became increasingly larger  with higher dose thresholds. In contrast, the remaining analyses of MOF revealed fracture risks that are comparable between DPP-4i users and GLP-1 RA users. The results of this study are in line with previous research and support the continued use of GLP-1 RAs in the management of T2D in patients at risk of fracture.https://www.dst.dk/da/TilSalg/Forskningsservice/Dataadgang.The datasets presented in this article are not readily available because. All eligible research organizations can apply for access to data at Statistics Denmark. Requests to access the datasets should be directed to All authors contributed to the article according to the ICJME requirements for co-authorship. All authors critically revised the paper for intellectual content and approved the submitted versions and the final version of the manuscript. ZKA, RV, and JS-L designed the study. ZKA, RV, JS-L and PV had access to all data used in the study. ZKA performed data management and statistical analyses with assistance from all co-authors. ZKA interpreted the data and wrote the manuscript. JS-L, RV, PV, RF-N and SG made ongoing critical revisions of study design and data interpretation.This work was supported by a Steno Collaborative Grant, Novo Nordisk Foundation, Denmark (Grant no. NNF18OC0052064).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aim of this cadaveric study was to evaluate the spread of an anaesthetic-contrast dye solution administered by an ultrasound-guided 1-point  or 2-point  TAP block technique. Contrast distribution was assessed by computed tomography whereas nerve staining was assessed by anatomical dissection. The 2-point-injection technique provided a wider injectate spread within the TAP than the 1-point technique. Further clinical studies are warranted to investigate the analgesic effect of these TAP block approaches in cats undergoing abdominal surgery.The p = 0.002). The prevalence of nerve staining was higher using TAP-SL than TAP-L (p = 0.001). The ventral branches of T10, T11, T12, T13, L1 and L2 were stained in 2/8, 2/8, 5/8, 7/8, 4/8 and 1/8, and in 7/8, 7/8, 8/8, 8/8, 8/8 and 1/8 using TAP-L and TAP-SL approaches, respectively. Computed tomography and dissection identified minimal injectate intraperitoneally or within the falciform ligament fat following 1 TAP-L and 2 TAP-SL. Ultrasound-guided TAP-SL provided better injectate distribution around the thoracolumbar spinal nerve branches than TAP-L.This study compared the distribution of a bupivacaine–iopamidol–dye solution following ultrasound-guided in-plane TAP injection using a 1-point (TAP-L) or 2-point (TAP-SL) approach in cat cadavers. Two cadavers were used to study the TAP sonoanatomy while eight cadavers were enrolled in a randomized, prospective, blinded investigation. Each cat randomly received a TAP-L with 0.5 mL/kg in one hemiabdomen and a TAP-SL with 0.25 mL/kg/point in the contralateral hemiabdomen. After injection, computed tomography and dissection were performed to assess contrast distribution and number of stained target nerves. TAP-SL resulted in a wider contrast spread (mm) compared with TAP-L (87 ± 7 versus 71 ± 9;  However, the TAP block does not cause pelvic limb motor impairment or urinary retention that can be observed after epidural administration of local anaesthetics and opioids.Locoregional anaesthesia is employed in a multimodal analgesic protocol to manage pain, decrease the consumption of systemic analgesic drugs  and their potential adverse effects, and potentially decrease the hospital length of stay . The ultritoneum ,4,5,6,7.ritoneum . An adeqmbar T9–L spinal ntransversus abdominis and obliquus internus abdominis performed at the midpoint between the last rib and the iliac crest using a lateral approach with a transversal needle orientation [transversus abdominis and rectus abdominis [In veterinary medicine, an US-guided TAP block was firstly described in a Canadian lynx as a bilateral 1-point injection of local anaesthetic within the mm. entation . The US-entation ,11. To oentation ,8,12. Nebdominis . Recentlbdominis . These sIn cats, a recent cadaveric study concluded that a 3-point TAP injection with a combination of subcostal and lateral-transversal TAP approaches is necessary to stain the thoracolumbar spinal nerve branches that provide sensory abdominal innervation . A cliniThe objective of this study was to compare the distribution pattern of 0.5 mL/kg of a solution administered by either a 1-point lateral-longitudinal TAP injection (TAP-L approach) or a 2-point subcostal and lateral–longitudinal TAP injection (TAP-SL approach) in cat cadavers using anatomical dissection and computed tomography.The hypothesis was that TAP-SL approach would result in a wider spread of injectate and staining of thoracolumbar spinal nerve branches compared with the TAP-L approach.This prospective, randomized, blinded, cadaveric study was approved by the Institutional animal care and use committee (Comité d’éthique de l’utilisation des animaux) of the Université de Montréal (no. 21-Rech-2111).A total of 10 frozen cadavers of adult cats  euthanized for reasons unrelated to this study were used. Exclusion criteria included body weight < 2 kg or >6 kg, body condition score > 7 or <3 on a scale from 1 to 9, and any evidence of skeletal and/or muscle injuries. Each cadaver was thawed at room temperature for 48 h prior to the experiment, and the thoracic and abdominal areas were clipped.The study was divided in two phases: gross anatomy and sonoanatomy study (phase I), and injectate spread evaluation and nerve staining after TAP injections by computed tomography (CT) and dissection (phase II).obliquus externus abdominis, the m. rectus abdominis, and the m. obliquus internus abdominis were dissected and reflected laterally to expose the nerves innervating the abdominal wall. Subsequently, dissections were performed to identify the thoracolumbar nerve branches up to their origin at the level of the intervertebral foramina. Afterwards, the contralateral hemiabdomen was sonographically evaluated and used to identify ultrasonographic landmarks for the TAP injection. Once the investigators (M.G. and S.M.) became familiar with the sonoanatomy, a 50 mm and 22-gauge spinal needle  was used to test an US-guided in-plane TAP subcostal and lateral approach (US settings described below). Two dye solutions, yellow and lime, were prepared using 0.5 mL of permanent tissue marking dye  diluted in 50 mL of 2% lidocaine  as previously described [rectus abdominis and transversus abdominis, and between the mm. transversus abdominis and obliquus internus abdominis, whereas the same volume of the lime-coloured solution was injected between the mm. obliquus internus abdominis and obliquus externus abdominis to identify the anatomical landmarks. Finally, the hemiabdomen was dissected to confirm injectate spread.Two cadavers were used to describe the relevant gross anatomy of the abdominal wall and spinal nerves (T9–L3). Dissections were performed on one hemiabdomen. The skin, the m. escribed . A volumEight cat cadavers were used to describe the US-guided injection of a bupivacaine–iopamidol–dye solution into the TAP by two approaches and evaluate the injectate spread and nerve staining by CT and dissection.In each cat, a 1-point TAP injection was performed on one hemiabdomen , whereas a 2-point TAP injection was performed on the contralateral hemiabdomen . The US setting was adjusted at the minimum depth (1.9 mm) while the gain was adjusted to optimize the image. A 22-gauge, 50 mm Quincke spinal needle  connected by a T-port  to a prefilled 5 mL syringe, was used to perform the TAP injections. All the injections were performed with the cat in dorsal recumbency by an anaesthesiologist familiar with US-guided TAP blocks (M.G.).obliquus internus abdominis could be identified between the mm. obliquus externus and transversus abdominis. The needle was introduced in-plane in a ventrocranial-to-dorsocaudal orientation and continually visualized until its tip was advanced into the TAP between the m. obliquus internus abdominis and the m. transversus abdominis , at 3–4 cm lateral to the abdominal midline and about 1 cm lateral to the mammary line, immediately caudal to the last rib . The trabdominis . Then, tlinea alba, the transducer was slid laterally and parallel to the costal arch and oblique to the midline . The CT images were obtained in a helical mode at 100 kVp, 300 mA, slice thickness 0.625 mm, and reconstructed using low-pass and high-pass filter algorithms. A board-certified radiologist (C.F.) who was blinded to the treatments provided image interpretation using the Horos™ software . The following CT features were recorded: total length (mm) of the contrast spread within the TAP in all directions , and any intra-abdominal contrast migration (yes/no). The predominant distribution patterns throughout the abdominal musculature for TAP-L and TAP-SL approaches were also described.linea alba. The aponeurosis of the m. obliquus externus abdominis was identified, dissected, and the belly of the muscle was reflected laterally to expose the aponeurosis of the m. obliquus internus abdominis. The m. rectus abdominis was detached from its lateral margin and dissected medially to expose the m. transversus abdominis and its aponeurosis. At this point, the cadaver was placed in lateral recumbency with the hemiabdomen to be dissected uppermost. The aponeurosis of the m. obliquus internus was dissected and reflected dorsally to expose the ventromedial branches of the thoracolumbar spinal nerves and the m. transversus abdominis.About 30 min after the end of CT examination, the cadavers were dissected to observe dye distribution and thoracolumbar spinal nerve ventral branch staining within the TAP. Two researchers (S.M. and M.G.) performed the dissections and the evaluation of the dye spread. Each cadaver was initially positioned in dorsal recumbency, and the skin was incised along the ventral midline from the mid-thorax to the pubis. The rectus sheath was dissected from the The ventromedial branches of the spinal nerves T10–L2 were considered the target nerves. Successful staining was defined as circumferential nerve staining with a length ≥ 1 cm . Locatiolinea alba, and the abdominal organs were carefully inspected to assess the presence of the bupivacaine–iopamidol–dye solution. Other complications such as presence of dye intramuscularly or in non-target fascial plane or in the abdominal cavity were recorded.After nerve staining evaluation, the cat was repositioned in dorsal recumbency, the abdominal cavity was exposed by incision of the t test. The number of ventral nerve branches successfully stained were compared with the Mann–Whitney U test, whereas any difference in the single nerve staining was compared with the Fisher’s exact test. Differences were considered statistically significant when p < 0.05.The number of cats enrolled in the study as well as the total volume of 1 mL/kg of injectate solution per cat, corresponding to 0.5 mL/kg of solution per hemiabdomen, were based on similar cadaveric studies in animals ,14,15. Stransversus abdominis extended through almost the entire abdominal cavity, ended at half of the width of the m. rectus abdominis and extended in a thin aponeurosis to the linea alba. The belly of the m. obliquus internus abdominis, which was found between m. transversus abdominis and the m. obliquus externus abdominis, ended at the mid-width of the hemiabdomen  and extended in a long aponeurosis to the rectus sheath. The m. obliquus externus abdominis is found along the lateral and ventral part of the abdomen, ending with the most superficial aponeurotic layer of the rectus sheath. Finally, the m. rectus abdominis extended longitudinally in the ventral abdominal wall from the external surface of the thorax to the pubic bone and it is enclosed in the rectus sheath, which multilayered aponeuroses were difficult to dissect. In one cat more than the other, a layer of adipose tissue was observed beneath and immediately caudal the costal arch, between the mm. rectus abdominis and transversus abdominis, extending laterally between the mm. obliquus externus and internus abdominis. The ventral branches of the spinal nerves T9–L3 were identified in both cadavers. These branches were further divided into ventrolateral and ventromedial branches. The ventrolateral branches were located at half of the width of thorax and abdomen  and further divided into terminal branches in the belly of m. rectus abdominis, and L3 in the belly of the m. transversus abdominis at the level of the pubis  body weight and body condition score were 3.0 (2.3–4.4) kg and 3.5 (2–5) in a scale from 1 to 9, respectively.A total of eight lateral injections by the TAP-L approach and eight lateral and eight subcostal injections by the TAP-SL approach were performed. Needle visualization was scored as “good” in all subcostal injections of the TAP-SL, “poor” in six TAP-L and six TAP-SL, and never “absent”.transversus abdominis. No intra-abdominal cavitary spread was observed with either approach.Spread of injectate was observed exclusively in the target plane following eight TAP-L and six TAP-SL. Following two subcostal injections in the TAP-SL approach, injectate spread was observed both dorsal and ventral to the m. transversus abdominis” and following 7 out of 16 lateral TAP injections (3 TAP-L and 4 TAP-SL) as “possible focal spread between the mm. obliquus internus and externus abdominis”. Focal (1.5 mm) intraperitoneal contrast solution was observed following two lateral TAP injections (1 TAP-L and 1 TAP-SL). In 2 TAP-SL, contrast solution was observed within the falciform ligament fat . No difference was found in the dorsal-to-ventral or medial-to-lateral spread of injectate between the two TAP approaches  compared with the TAP-L . The total number of target nerves stained with the TAP-SL approach was 39 out of 48 (81.3%) and was significantly higher than the number of target nerves stained with the TAP-L approach, which was 21 out of 48 (43.8%). When analysing individual nerve staining by both approaches, the TAP-SL resulted in a significantly higher success rate of staining T10 and T11 compared with the TAP-L (p = 0.04). No significant difference was observed between the two approaches in staining of nerves caudal to T12. The nerve L2 was stained only in one out of eight injections by either approach. The ventromedial branches of T9 and L3 were not stained by any injection.In all cadavers, the ventromedial branches of the T10–L3 spinal nerves were identified within the TAP. The ventromedial branch of T9 was identified within the TAP in 12 out of 16 hemiabdomens at the level of the xiphoid process. The ventromedial branch of L3 reached the ventral midline at the level of the pubis in one cat. The ventromedial branch of the T10–L2 spinal nerves successfully stained by TAP-L and TAP-SL is summarized in This study described the anatomical and sonographic findings of the TAP in cats. Injectate distribution and consistency of nerve staining was also assessed by ultrasonography, CT and dissection after a TAP injection using two approaches. This information allows better understanding of potential local anaesthetic distribution, complications and limitations of this locoregional technique in the clinical setting.In this study, a significant difference in cranial-to-caudal injectate spread was found between the 1-point TAP-L approach and the 2-point TAP-SL approach. Nevertheless, the most cranial and caudal margins of the solution spread corresponded to the nerve T10 and L2, with either approach. On the other hand, the TAP-SL approach resulted in a more consistent staining of the target nerves than TAP-L, which could represent a more predictable distribution of an anaesthetic solution in cats.The ventromedial branch of L2 was stained only in 12.5% of cases after TAP-SL and TAP-L. Therefore, the caudal portion of the abdominal wall could possibly not be desensitized, depending on the incision size, by a local anaesthetic. The lateral TAP injection was performed inserting the needle immediately caudal to the last ribs at 3–4 cm from the ventral midline; it is possible that a more caudal injection point  would lead to a better caudal injectate distribution and consistent staining of L2 than the present technique. Further anatomical studies are warranted to confirm or reject this hypothesis.The ventromedial branch of T10, T11, T12, T13, L1 and L2 was stained in 87.5%, 87.5%, 100%, 100%, 100% and 12.5% of cases with our lateral-longitudinal TAP-SL approach when compared with 57.1%, 100%, 87.5%, 28.5%, 42.8% and 87.5%, respectively, by a previously reported lateral-transversal TAP-SL approach [transversus abdominis, whereas in one out of eight cadavers, it reached the midline at the level of the pubis. Neither of the TAP approaches employed in this study stained the ventral branches of T9 and L3. These findings demonstrate that the efficacy of a TAP block may vary due to anatomical individual differences independently of similar volumes of administration and techniques. It is possible that the efficacy of a TAP block may also vary according to the extent (size) and location  of a laparotomy.Individual variability in the pathway of the thoracolumbar nerve branches in the TAP was observed as reported in similar cadaveric studies in cats  and in dtransversus abdominis and suspected presence of contrast between the mm. obliquus internus and externus abdominis were observed in the CT. During gross anatomical dissection, the m. obliquus internus abdominis and sometimes the m. obliquus externus abdominis were partially stained by the dye solution, as previously reported [In this study, the ventrolateral branches of the thoracolumbar nerves responsible for the abdominal cutaneous dermatome innervation were also stained. This phenomenon was also observed in a previous study ; howeverreported . Diffusitransversus abdominis was observed with the US. Nevertheless, gentle repositioning of the needle tip under US visualization resulted in partial intramuscular injection in the m. rectus abdominis, suggesting that the bevel of the needle was larger than the TAP and the m. transversus abdominis. Analysis of CT images also revealed focal intraperitoneal injections that were not observed with real-time US visualization and could not be identified by gross dissection following two lateral TAP injections. These two focal infiltrations of contrast solution could be explained by puncture of the peritoneum with the needle tip, without evident needle perforation of the abdominal cavity. The Quincke needle used in this study has a 20-degree-long cutting bevel, and the m. transversus abdominis of the three cats, in which these complications were observed, was about 0.2 mm thick. It is possible that a different type of needle with a shorter bevel  would be more appropriate to perform TAP injections in cats.Although no bupivacaine–iopamidol–dye solution was observed free in the abdominal cavity, a total of 4 out of 24 injections (16.7%) resulted in intraperitoneal spread of injectate solution. Both CT and dissection revealed the presence of injectate solution into the falciform ligament following two subcostal injections. In these two cases, distribution of injectate solution both in the TAP and ventral to the m. The prevalence of intraperitoneal injection reported herein (16.7%) was lower than that observed in similar investigations into dog (23%) and human cadavers (25%) using CT studies ,18. TherThe injectate volume used was based on a previous cadaveric study in cats . MoreoveThe findings obtained by cadaveric studies should be carefully applied to a clinical setting. The physical properties of the bupivacaine–iopamidol–dye solution employed in this investigation differ from local anaesthetics, and it is possible that this might have influenced the results. The contrast was diluted to a proportion of 1:10 as previously described in cadaveric studies in humans and dogs ,18. A fuThis investigation has limitations, and some have already been discussed. The injectate distribution in a cadaver model may differ from the distribution encountered in live animals because of tissue modifications related to freezing and thawing, systemic drug absorption according to the tissue temperature, and changes in abdominal pressure with breathing. A further limitation is that one researcher performing the dissections was not blinded, which may have biased the interpretation of results. Finally, it is unknown how the results and potential complications would have been changed if the TAP block had been performed by an inexperienced veterinarian.The 2-point TAP-SL approach resulted in better injectate distribution than the 1-point TAP-L approach. Although the spinal nerve L2 was rarely stained by either approach, the 2-point TAP injections consistently stained all ventral branches of spinal nerves T10–L1, suggesting that the TAP-SL approach could be superior to the TAP-L approach.Investigation of the clinical efficacy of an anaesthetic solution injected by a combined subcostal and lateral-longitudinal TAP approach in cats is currently ongoing.